Characterization of the Oxygen Heterocyclic Compounds (Coumarins, Psoralens, and Polymethoxylated Flavones) in Food Products Mariosimone Zoccali1, Adriana Arigò1, Marina Russo2, Francesca Rigano3, Paola Dugo1,2,3 and Luigi Mondello1,2,3 1Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, University of Messina, Italy 2University Campus Bio-Medico of Rome, Italy 3Chromaleont s.r.l. University of Messina, Italy Italy Universityof Messina, [email protected] Focus on the Oxygenated Heterocyclic Compounds secondary metabolites commonly found in all citrus plants Furocoumarin or Coumarin psoralen Polymethoxyflavone Beneficial effect on humans: Harmful effect on humans: ➢Anticancer ➢Phototoxic ➢Antioxidant ➢Inhibitor of intestinal and liver drug Italy Universityof Messina, ➢Antibacterial metabolism ➢Anti-inflammatory Development of high sensitive analytical methods Isolation of pure compounds Citrus essential oils Citrus bergamia Risso Food industry Cosmetic industry University of Messina, Italy Universityof Messina, Flavour and fragrance industry Opinion on Coumarin in Food products The Panel considered the toxicity studies and the studies on the metabolism of coumarin in humans with CYP2A6 polymorphism that have become available since the last opinion of 2004, as well as clinical studies, and concluded to maintain the TDI of 0.1 mg coumarin/kg bw allocated in the 2004 opinion. University of Messina, Italy Universityof Messina, Furocoumarins in Food products Furocoumarins are a class of photoactive compounds found in several plant species and may be responsible for the observed association between consumption of citrus products and the risk of skin cancer. When irradiated with UV light, furocoumarins can undergo photoactivation, putting them into an excited and highly reactive triplet state. In this state, certain Italy Universityof Messina, furocoumarins can form adducts with DNA, induce protein denaturation, form cycloadducts with saturated fatty acids, and react with ground state oxygen to form reactive oxygen species that can cause cellular damage Analytical techniques employed for the characterization of the oxygenated heterocyclic compounds Chromatography Detection/identification TLC NP-LC Fluorescence RP-LC EI-MS LC×LC APCI-MS Micro-LC PDA/UV Nano-LC LRI SFC Comprehensive two dimensional separation of oxygen LC) University of Messina, Italy Universityof Messina, - 5 heterocyclic components of lemon oil 2 81APCI- (P. Dugo, M. Ramírez Fernández, A. Cotroneo, G. 4 MS 6 Dugo, L. Mondello. J. Chromatogr. Sci. 44, (2006) 561) PDA/U 3 7 V 7 9 1 RF 11 Reversed phase (RP Reversedphase 9 10 The elution order in both dimensions represents a 6 EI-MS key information for the identification of unknown 10 11 in the 2D plot Normal phase (NP-LC) Identification strategies 1 The UV spectrum easily allows identifying the chemical class (coumarin, furocoumarin or polymethoxyflavones), as well as the position of substituents in the heterocyclic structure 2 The interpretation of MS spectra can lead to a more reliable identification 3 Both UV and MS spectra can be included in UV and MS libraries to make the identification process automatic and faster 4 Retention time information can be used complementarily to spectral data University of Messina, Italy Universityof Messina, UV library IUPAC NAME CAS University of Messina, Italy Universityof Messina, UV library: automatic identification University of Messina, Italy Universityof Messina, Both UV and retention time are necessary to confirm the identification. MS library University of Messina, Italy Universityof Messina, Usually, only the molecular weight information is provided (impossibility to identify isomers) Interferences from the matrix or a matrix effect can significantly reduce the spectral similarity (low identification reliability) mAU 200 195 196 195 201 202 200 221 222 222 The use The use of alibrary MS help the identification of species characterized by 250 254 254 254 268 269 270 300 287 288 289 313 314 314 350 nm Byakangelicol Phellopterin Byakangelicin identical UV spectrum MS library University of Messina, Italy Remarks/Evaluations In the case of molecules characterized by the same molecular weight, more powerful MS instrumentation (high resolution, tandem MS systems) would be necessary for structure elucidation. One of the main purpose of the present research is the finding of less expensive and easier solution to improve identification reliability of LC methods University of Messina, Italy Universityof Messina, Linear retention index (LRI) approach More recent approaches Identification tools in chromatography Retention behaviour Spectral information LC GC GC LC No universal Stable LRI system EI-MS spectra API-MS and/or LRI system by Van Den Dool UV spectra and Kratz* University of Messina, Italy Universityof Messina, HIGHLY RELIABLE IDENTIFICATION POOR IDENTIFICATION POWER *Van Den Dool and Kratz , J. Chrom. A, 11 (1963) 436-471 Use of LRI as additional filter • In gas chromatography LRI represents a system in which the retention times of analytes are correlated to a reference standard mixture, making retention data dependent only on the three terms interaction analyte-stationary phase-mobile phase and indipendent from other chromatographic conditions (column dimension, temperature program, mobile phase linear velocity) • LRI are normally employed as an extra criterion of mass spectral library searching: compounds with a high spectra matching but with a LRI value falling out from a selected range are automatically excluded from the list of possible candidates • In liquid chromatography retention data are strongly dependent from the mobile Italy Universityof Messina, phase composition, thus increasing the number of parameters to be considered. • The building of an LRI database would be more meaningful in the LC-MS system where retention data might be complementary to the identification capability of MS, as in GC-MS. LRI in LC A consistent literature refers about the employment of retention index during the decades ’80s-90s, sometimes combined with UV spectral information for identification purposes. An example Bogusz and Wu, J. Anal. Toxicol. 1991, 15, 188-197. The influence of several experimental conditions, such as the stationary phase chemistry or mobile phase composition, was evaluated, allowing to conclude that a standardization of the LC conditions is necessary to create a usable database. Bogusz et al. Italy Universityof Messina, «The elution conditions should be carefully standardized in order to obtain reproducible results» J. Liq. Chromatogr. R.T. 1996, 19, 1291-1316. In the last decades, the higher batch-to-batch reproducibility in LC columns and instrumentation can lead to a more reliable and stable LRI system, also at interlaboratory levels. Reference standard mixture “many attempts” 1-nitroalkanes Three homologue series have been reported in literature: alkan-2-ones from C3 to C23 (low UV absorption) Analytes alkyl aryl ketones from acetophenone to heptanophenone (long elution times) 1-nitroalkanes from nitromethane to 1-nitrooctane (low availibility) Alkyl aryl ketones Italy Universityof Messina, LRI calculation Bogusz et al. J. Anal. Toxicol. 1988, 12, 325-329 LC-MS/MS approach as a novel unified tool for the quali-quantitative characterization of oxygen heterocyclic compounds Comparison with the recent LC-PDA method University of Messina, Italy Universityof Messina, Project schedule (work-flow) Nexera-i Nexera X2 UHPLC LCMS-8060 ▪ Validation of the LC-PDA ▪ Validation of the LC-MS/MS method (LoD, LoQ, repeatability, accuracy, method (LoD, LoQ, repeatability, accuracy, linearity) linearity) ▪ Creation of an UV-library ▪ Optimization of MRM transition parameters for each target compouds ▪ Calculation of the LRI of each target compounds ▪ Creation of MS and MS/MS-libraries ▪ Identification of oxygenated ▪ Calculation of the LRI of each target heterocyclic compounds through compounds both UV library and LRI ▪ Identification of oxygenated ▪ Quantitative determination of targets heterocyclic compounds through Italy Universityof Messina, compounds in the real samples by means of both MS and MS/MS libraries and LRI calibration curves. ▪ Realization of calibration curves for each STD compounds ▪ Quantitative determination of targets compounds in the real samples by means of calibration curves Analysis conditions Column: Ascentis Express C18 (50 × 4.6 mm, 2.7 mm) Solvent A: Water/Methanol/THF 85:10:5 v/v) Solvent B: Methanol/THF 95:5 v/v) Gradient: Flow rate: 2 mL/min Oven Temperature: 40° C PDA parameters Time constant: 0.48 sec Sampling: 4.1667 Hz Range: 190-370 MS parameters Interface: APCI positive MRM mode University of Messina, Italy Universityof Messina, Nebulizing Gas Flow: 3 L/min Interface Temperature: 450° C DL Temperature: 300° C Heat Block Temperature: 300° C Drying Gas Flow: 15 L/min CID GAS: 270 kPa LC-PDA experimental work-flow Inject the sample solution Inject the reference homologue series for LRI calculation Identify the sample components by OHCs library using UV and LRI filters University of Messina, Italy Universityof Messina, Quantify the sample by external calibration UHPLC-PDA (iseries) instrumentation used in this study PDA chromatogram of all the target analytes mAU 315nm 10 20 3 24+25 12 26+27 22 23 15 2 11 28 29 33 35 1 5 20 34 8 9 13+14 31 10 6 15 21 4 7 16 18 32 5 17 19 30 0 University of Messina, Italy Universityof Messina, 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 min Mix of all STDs (35 compounds among